Method and apparatus for automated staining of biological materials

ABSTRACT

Various embodiments of the present invention provide methods and systems for optimally staining biological samples for analysis. In one embodiment, images of a sample are recorded before and after the application of a staining reagent and a decolorization reagent. A difference image is then generated based at least in part on a comparison of the images of the sample recorded before and after the application of the staining and decolorization reagents. Application parameters of the staining and decolorization reagents are then corrected based at least in part on the difference image such that the reagents may be optimally reapplied to generate a final image of the sample that may enable a user to better differentiate at least one target of interest in the sample. In one example, embodiments of the present invention allow for the generation of images that show only Gram-positive bacteria or only Gram-negative bacteria.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/529,065, filed on Jun. 3, 2010, which is anational stage application filed under 35 U.S.C. 371 of InternationalApplication No. PCT/US2008/055471, filed Feb. 29, 2008, which claimspriority from U.S. Provisional Application No. 60/892,736, filed Mar. 2,2007, each of which is incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

The present invention relates to methods and apparatus for the stainingof biological material for the purpose of detecting and identifyingdisease-causing microorganisms. More particularly, the present inventionrelates to methods and apparatus for performing automated Gram stains onmicroscope slides.

BACKGROUND OF THE INVENTION

Gram staining is one of the most frequently performed procedures inmodern microbiology laboratories. Such procedures are used to broadlyclassify bacteria as “Gram-positive” or “Gram-negative.” After affixinga bacteria-containing sample to a microscope slide, the sample istreated using staining reagents such as crystal violet in combinationwith Gram's iodine. This first step stains all bacteria a deep blue orviolet. The principal difference between “Gram-positive” and“Gram-negative” bacteria is that in “Gram-positive” samples, thestaining reagents are absorbed within the whole cellular structure,while in the “Gram-negative” samples, staining occurs onlysuperficially. Consequently, when the sample is subsequently treatedwith a decolorizing agent (such as acid alcohol), Gram-negative bacteriatend to lose their color, while Gram-positive bacteria remain stainedblue or violet.

Gram stains are conventionally prepared and analyzed manually. ManualGram staining is labor-intensive, requires skilled personnel, and mayfail to achieve optimum staining characteristics in the sample set.Non-optimum staining may result in false Gram-positives as well as falseGram-negatives.

There have been different attempts to automate the Gram stainingprocedure. See, for example, U.S. Pat. No. 4,029,470 to Wilkins et al.(“Wilkins”). Similarly, the Aerospray Slide Stainer, availablecommercially from Wescor, Inc. of Logan, Utah is a rudimentary automatedstaining apparatus. Other staining devices include the Midas III SlideStainer, commercially available from Merck KGaA of Darmstadt, Germany;the Poly Stainer, which is commercially available from IUL InstrumentsGmbH of Koenigswinter, Germany; and the Automated Gram Stainer,commercially available from the GG&B Company of Wichita Falls, Tex. anddescribed generally in U.S. Pat. No. 6,468,764 to Gibbs et al. None ofthese instruments or techniques, however, allow for the capability ofapplying staining and/or decolorizing agents before, during, and/orafter examination and/or image processing of a sample slide.

Consequently, there exists a need for an improved automated stainingmethod and system.

SUMMARY OF THE INVENTION

The present invention, in various embodiments, provides a method andsystem that overcomes many technical problems with regard to theoptimization of staining procedures (such as Gram staining, for example)for biological samples. Specifically in one embodiment, the inventionprovides a method for staining a sample, comprising steps for operablyengaging a sample with a sample stage of an imaging system, andrecording images of the sample using the imaging system before and afteran application of a staining reagent and a decolorization reagent to thesample. The method also comprises generating a difference image based atleast in part on a comparison of the images of the sample recordedbefore and after the application of the staining reagent and thedecolorization reagent to the sample, and correcting the application ofthe staining reagent and the decolorization reagent based at least inpart on the difference image. The method further comprises reapplying atleast one of the staining reagent and the decolorization reagentaccording to the correcting step and generating a final image of thesample so as to differentiate at least one target of interest in thesample that may be rendered discernible by the reapplying step.

Some embodiments further provide a method for performing a stainingprocedure for a sample comprising steps for recording a first image ofthe sample and applying a staining agent to the sample so as to preparea stained sample comprising a plurality of stained entities. Some methodembodiments further comprise steps for washing the sample with a washingsolution prior to recording the first image of the sample so as toensure that each sample is washed to establish a “zero” level ofstaining prior to recording the first image (which provides a basis forcomparison with images recorded of the sample after the application ofat least one of the staining agent and the decolorization agent).

The plurality of stained entities may include at least one of aplurality of stained entities and a plurality of superficially stainedentities. In some such embodiments, the staining agent may comprise aGram stain such that the superficially stained entities, if present,comprise a plurality of Gram-negative entities, and such that thestained entities, if present, comprise a plurality of Gram-positiveentities. The method may further comprise recording a second image ofthe stained sample and generating a first difference image by comparingthe second image to the first image so as to determine a location of atleast one of the stained entities on the surface.

The method may further comprise applying a decolorizing agent to thestained sample so as to prepare a partially decolorized sample whereinat least a portion of the staining reagent is removed from thesuperficially stained entities, and recording a third image of thepartially decolorized sample. The method may also comprise generating asecond difference image by comparing the third image to the second imageso as to determine a location of at least one of the superficiallystained entities on the surface. Finally, the method may also compriseanalyzing the second difference image to determine an exposure timeduring which the decolorizing agent could be applied to the partiallydecolorized sample to substantially decolorize the superficially stainedentities without substantially decolorizing the stained entities.

In some embodiments, the method may further comprise applying thedecolorizing agent to the partially decolorized sample for thedetermined exposure time so as to prepare a substantially decolorizedsample wherein the superficially stained entities are substantiallydecolorized and wherein the stained entities are not substantiallydecolorized. In some such embodiments, the method may further comprise:(1) recording a fourth image of the substantially decolorized sample;(2) generating a third difference image by comparing the fourth image tothe second image, wherein the third difference image depicts thelocation of at least one of the superficially stained entities on thesurface; and (3) generating a fourth difference image by comparing thefourth image to the first image, wherein the fourth difference depicts alocation of at least one of the stained entities on the surface. Inmethod embodiments wherein the locations of the stained entities and thesuperficially stained entities are at least partially discernible in atleast one of the third and fourth difference images, the method mayfurther comprise analyzing a morphology of the stained entities and/orthe superficially stained entities.

Various embodiments of the present invention may also provide systemsfor performing an optimized staining procedure for a sample. In oneembodiment, the system comprises a flow chamber defining channel influid communication with a supply of a staining agent and a supply of adecolorizing agent. Furthermore, in some such embodiments, the flowchamber may comprise a surface configured for operably engaging thesample therewith. The system may also comprise a fluidics system influid communication with the flow chamber, the supply of the stainingagent, and/or the supply of the decolorizing agent.

According to such system embodiments, the fluidics system (incooperation with the flow chamber, for example) may be configured forapplying the staining agent to the sample so as to prepare a stainedsample comprising a plurality of stained entities, wherein the pluralityof stained entities may include at least one of a plurality of stainedentities and a plurality of superficially stained entities. As describedabove with respect to various method embodiments of the presentinvention, the staining agent may comprise a Gram stain such that thesuperficially stained entities, if present, comprise a plurality ofGram-negative entities, and such that the stained entities, if present,comprise a plurality of Gram-positive entities. According to varioussystem embodiments, the fluidics system (in cooperation with the flowchamber, for example) may be further configured for applying thedecolorizing agent to the stained sample so as to prepare a partiallydecolorized sample wherein at least a portion of the stain is removedfrom the superficially stained entities.

In various system embodiments, the flow chamber may be adapted forreceiving a slide defining the surface for operably engaging the sampletherewith. According to such embodiments, the flow chamber may furthercomprise a flow channel housing defining a slide aperture configured forreceiving the slide such that the sample is disposed substantiallybetween the slide and the flow channel housing when the slide isdisposed in the slide aperture. Furthermore, the flow channel housingmay comprise a substantially translucent material such that the imagingsystem is capable of recording images of the sample while the sample isdisposed between the slide and the flow channel housing.

The system may also comprise an imaging system disposed adjacent to theflow chamber such that the sample is positioned within a field of viewof the imaging system. In such system embodiments, the imaging systemmay be configured for monitoring an application of at least one of thestaining agent and the decolorizing agent via the flow chamber.Furthermore, the imaging system may also be configured for generating adifference image by comparing at least one image of the sample obtainedbefore a first application of the decolorizing agent and at least oneimage of the sample obtained after the first application of thedecolorizing agent. The imaging system may also be configured to becapable of determining an exposure time for a second application of thedecolorizing agent based at least in part on the difference image so asto substantially decolorize the superficially stained entities withoutsubstantially decolorizing the stained entities such that the stainedentities may be more readily discerned from the superficially stainedentities.

The system may also comprise, in some embodiments, a controller devicein communication with the imaging system and the fluidics system. Thecontroller device may be further configured to control the applicationof at least one of the staining agent and the decolorizing agent basedat least in part on the images and/or difference images generated by theimaging system.

In some system embodiments, the imaging system may comprise a cameradevice configured to be capable of recording images of the sample whilethe sample is disposed in the flow chamber (such as between the slideand the flow channel housing, for example). According to some suchembodiments, the imaging system may also comprise an actuator deviceoperably engaged with the camera device and configured for adjusting aposition of the camera device relative to the flow chamber. Furthermore,in some system embodiments, the imaging system may also comprise animage processing computer configured for generating the differenceimage. In some such embodiments, the image processing computer may beconfigured for generating the difference image using processes that mayinclude, but are not limited to: image subtraction; image addition;image ratio calculation; and combinations of such processes.

As described generally herein with respect to various methodembodiments, the imaging system may be configured for recording a firstimage of the sample and subsequently recording a second image of thestained sample after applying the staining agent to the sample using thefluidics system in cooperation with the flow chamber. The imaging systemmay be further configured for generating a first difference image bycomparing the second image to the first image so as to determine alocation of at least one of the stained entities (i.e. the“Gram-positive” entities, in some embodiments) on the surface.Furthermore, the imaging system may be further configured for recordinga third image of the partially decolorized sample after applying thedecolorizing agent using the flow chamber, and subsequently generating asecond difference image by comparing the third image to the second imageso as to determine a location of at least one of the superficiallystained entities (i.e. the “Gram-negative” entities, in someembodiments). The imaging system may be further configured for analyzingthe second difference image to determine an exposure time during whichthe decolorizing agent should be applied to the partially decolorizedsample to substantially decolorize the superficially stained entitieswithout substantially decolorizing the stained entities. As describedherein, in some such system embodiments, the flow chamber may be furtherconfigured for applying the decolorizing agent to the partiallydecolorized sample for the determined exposure time so as to prepare asubstantially decolorized sample wherein the superficially stainedentities, if present, are substantially decolorized and wherein thestained entities, if present, are not substantially decolorized.

In some such system embodiments, the imaging system may be furtherconfigured for recording a fourth image of the substantially decolorizedsample and subsequently generating a third difference image by comparingthe fourth image to the second image. The third difference image maydepict the location of at least one of the superficially stainedentities. The imaging system may also be configured for generating afourth difference image by comparing the fourth image to the firstimage, wherein the fourth difference depicts a location of at least oneof the stained entities.

Thus the methods and systems for performing staining procedures forbiological materials, as described in the embodiments of the presentinvention, provide many advantages that may include, but are not limitedto: providing methods and systems that are capable of monitoringdiscernible optical changes in a sample during a staining process so asto better control and/or optimize the staining and decolorization of thesample; providing methods and systems that may be especially capable ofclassifying and locating Gram-stained entities within a sample bymonitoring optical changes that may be discernible between stainingand/or decolorization steps and highlighting those optical changes thatare unique to Gram-positive and/or Gram-negative entities within thesample; providing systems and methods that may be uniquely capable ofminimizing “false positives” and/or “false negatives” when analyzingstained samples in search of entities of interest within the sample(such as disease-causing bacteria, for example); and providing anoptimized staining method and system that may be monitored and/orcontrolled in real time by an optical imaging apparatus. Thus, variousembodiments of the present invention may also provide an “all-in-one”staining apparatus and sample analysis tool wherein finished (andoptimized) stained sample slides may remain on the same imagingapparatus for downstream morphological investigations. This completeintegration of the sample preparation (i.e. staining) steps and analysissteps may allow for more efficient laboratory throughput.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily drawn toscale, and wherein:

FIGS. 1-10 show a series of “single-line” images recorded of a samplecontaining both stained (i.e. Gram-positive, for example) andsuperficially stained (i.e. Gram-negative, for example) entities,according to one embodiment of the present invention;

FIG. 1 shows a non-limiting depiction of a “single line” image of anunstained sample, corresponding to a first image, produced according toone embodiment of the present invention;

FIG. 2 shows a non-limiting schematic of a “single line” of a sample,showing the locations of stained entities (i.e. Gram-positive entities)in the sample, according to one embodiment of the present invention;

FIG. 3 shows a non-limiting schematic of a “single line” of a sample,showing the locations of superficially stained entities (i.e.Gram-negative entities) in the sample, according to one embodiment ofthe present invention;

FIG. 4 shows a non-limiting depiction of a “single line” image of astained sample, corresponding to a second image, produced according toone embodiment of the present invention;

FIG. 5 shows a non-limiting depiction of a “single line” image of asample, corresponding to a first difference image, produced according toone embodiment of the present invention, highlighting the locations ofstained and superficially stained entities in the sample;

FIG. 6 shows a non-limiting depiction of a “single line” image of asample, corresponding to a third image, produced according to oneembodiment of the present invention after a 35% decolorization of thesample;

FIG. 7 shows a non-limiting depiction of a “single line” image of asample, corresponding to a second difference image, produced accordingto one embodiment of the present invention, highlighting the effect ofthe 35% decolorization on superficially stained entities in the sample;

FIG. 8 shows a non-limiting depiction of a “single line” image of asample, corresponding to a fourth image, produced according to oneembodiment of the present invention, after a 100% decolorization of thesample;

FIG. 9 shows a non-limiting depiction of a “single line” image of asample, corresponding to a third difference image, produced according toone embodiment of the present invention, highlighting the locations ofsuperficially stained (i.e. Gram-negative, for example) entities in thesample;

FIG. 10 shows a non-limiting depiction of a “single line” image of asample, corresponding to a fourth difference image, produced accordingto one embodiment of the present invention, highlighting the locationsof stained entities (i.e. Gram-positive, for example) in the sample;

FIGS. 11-19 show a series of “single-line” images recorded of a samplecontaining only stained (i.e. Gram-positive, for example) entities,according to one embodiment of the present invention;

FIG. 11 shows a non-limiting depiction of a “single line” image of anunstained sample, corresponding to a first image, produced according toone embodiment of the present invention;

FIG. 12 shows a non-limiting schematic of a “single line” of a sample,showing the locations of stained entities (i.e. Gram-positive entities)in the sample, according to one embodiment of the present invention;

FIG. 13 shows a non-limiting schematic of a “single line” of a sample,showing the lack of superficially stained entities (i.e. Gram-negativeentities) in the sample, according to one embodiment of the presentinvention;

FIG. 14 shows a non-limiting depiction of a “single line” image of astained sample, corresponding to a second image, produced according toone embodiment of the present invention;

FIG. 15 shows a non-limiting depiction of a “single line” image of asample, corresponding to a first difference image, produced according toone embodiment of the present invention, highlighting the locations ofstained entities in the sample;

FIG. 16 shows a non-limiting depiction of a “single line” image of asample, corresponding to a third image, produced according to oneembodiment of the present invention after a 35% decolorization of thesample;

FIG. 17 shows a non-limiting depiction of a “single line” image of asample, corresponding to a second difference image, produced accordingto one embodiment of the present invention, further depicting the lackof superficially stained entities in the sample;

FIG. 18 shows a non-limiting depiction of a “single line” image of asample, corresponding to a third difference image, produced according toone embodiment of the present invention, highlighting the lack ofsuperficially stained (i.e. Gram-negative, for example) entities in thesample;

FIG. 19 shows a non-limiting depiction of a “single line” image of asample, corresponding to a fourth difference image, produced accordingto one embodiment of the present invention, highlighting the locationsof stained entities (i.e. Gram-positive, for example) in the sample;

FIGS. 20-29 show a series of “single-line” images recorded of a samplecontaining only superficially-stained (i.e. Gram-negative, for example)entities, according to one embodiment of the present invention;

FIG. 20 shows a non-limiting depiction of a “single line” image of anunstained sample, corresponding to a first image, produced according toone embodiment of the present invention;

FIG. 21 shows a non-limiting schematic of a “single line” of a sample,showing the lack of stained entities (i.e. Gram-positive entities) inthe sample, according to one embodiment of the present invention;

FIG. 22 shows a non-limiting schematic of a “single line” of a sample,showing the locations of superficially stained entities (i.e.Gram-negative entities) in the sample, according to one embodiment ofthe present invention;

FIG. 23 shows a non-limiting depiction of a “single line” image of astained sample, corresponding to a second image, produced according toone embodiment of the present invention;

FIG. 24 shows a non-limiting depiction of a “single line” image of asample, corresponding to a first difference image, produced according toone embodiment of the present invention, highlighting the locations ofsuperficially stained entities in the sample;

FIG. 25 shows a non-limiting depiction of a “single line” image of asample, corresponding to a third image, produced according to oneembodiment of the present invention after a 35% decolorization of thesample;

FIG. 26 shows a non-limiting depiction of a “single line” image of asample, corresponding to a second difference image, produced accordingto one embodiment of the present invention, highlighting the effect ofthe 35% decolorization on superficially stained entities in the sample;

FIG. 27 shows a non-limiting depiction of a “single line” image of asample, corresponding to a fourth image, produced according to oneembodiment of the present invention, after a 100% decolorization of thesample;

FIG. 28 shows a non-limiting depiction of a “single line” image of asample, corresponding to a third difference image, produced according toone embodiment of the present invention, highlighting the locations ofsuperficially stained (i.e. Gram-negative, for example) entities in thesample;

FIG. 29 shows a non-limiting depiction of a “single line” image of asample, corresponding to a fourth difference image, produced accordingto one embodiment of the present invention, highlighting the lack ofstained entities (i.e. Gram-positive, for example) in the sample;

FIG. 30 shows a non-limiting flow chart summarizing method steps forperforming a staining procedure for a sample, according to oneembodiment of the present invention;

FIG. 31 is a non-limiting schematic depiction of a system according toone embodiment of the present invention, showing an imaging systemdisposed adjacent to a flow chamber configured for applying a stainingagent and/or a decolorizing agent to a sample;

FIG. 32 is a non-limiting schematic depiction of a system according toone embodiment of the present invention, including a flow channel and animaging system including a camera device, an actuator device, an imageprocessing computer, and a systems controller;

FIG. 33 shows several non-limiting views of a flow chamber, according toone embodiment of the present invention, the flow chamber including aslide defining the surface for operably engaging the sample therewithand a flow channel housing defining a slide aperture configured forreceiving the slide;

FIG. 34 shows several non-limiting views of a flow chamber, according toone embodiment of the present invention, showing steps for operablyengaging a slide with a flow channel housing of the flow chamber;

FIG. 35 shows a non-limiting schematic of a flow chamber and an imagingsystem disposed adjacent to the flow chamber, according to oneembodiment of the present invention; and

FIG. 36 shows a non-limiting flow chart summarizing method steps for amethod for optimally staining a sample, according to one embodiment ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

While the various method and system embodiments disclosed herein aredescribed in the context of a Gram staining procedure, it should beunderstood that the various method and system embodiments describedherein may also be applied to many other staining procedures that maycause optical and/or colorimetric changes in a sample. Furthermore, itshould be emphasized that the exemplary images shown in FIGS. 1-29represent only one line of a two-dimensional image. In reducing thevarious method and system embodiments of the present invention topractice one may deal with many such lines, combined intotwo-dimensional images of a sample operably engaged with a slide, forexample. Also, as described further herein, in some embodiments, a usermay utilize the imaging system (and/or a camera device 8 includedtherein) to record images in more than one field on the sample slide tolocate and classify multiple bacteria or other stained and superficiallystained entities in the sample.

It should also be understood that the terms “recording images” as usedherein are not limited to analog imagery captured by a camera, forexample. More particularly, in some embodiments, “recording images” mayrefer to the capture of data that is indicative of an image and/orcorresponds to an image. For example, “recording images” may comprise,in some embodiments, collecting digital data corresponding to colorand/or light intensity at one or more points on a surface.

It should be understood that various method and system embodiments ofthe present invention take advantage of the fact that staining processes(such as Gram staining, for example) may produce discernible and/ordetectable optical changes in a sample. By monitoring such changesduring the staining process directly on a surface (such as a microscopicslide) with which the sample is operably engaged, the steps of stainingand subsequent decolorization can be controlled and optimized. Thisaspect of the present invention may apply, in particular, todecolorization steps. For example, it is known that in Gram stainingprocesses, too little decolorization may yield false Gram-positives, andthat too much decolorization may yield false Gram-negatives. Asdescribed herein, various embodiments of the present invention may aidin improving the degree of decolorization in a variety of stainingprocesses (including, for example, Gram staining processes).

It should be further understood that various embodiments of the presentinvention may also utilize difference images (see, for example, FIGS. 5,7, 9, 10) to highlight discernible and/or detectable optical changesthat may occur differently for Gram-positive entities and Gram-negativeentities in the sample, allowing for classification of these entities(which may include, but are not limited to, Gram-stained bacterialentities). As described further herein, various system and methodembodiments of the present invention may allow for the accuratedetermination of specific locations for Gram-positive bacteria, ifpresent, and/or Gram-negative bacteria on the surface with which asample is operably engaged (such as a typical microscope slide) byanalyzing, for example, various difference images generated by comparingimages of the slide taken before and after a partial decolorization step(see step 107, FIG. 30, for example) and/or before and after a completedecolorization step (see step 112, FIG. 30, for example).

As shown generally in FIG. 30, various embodiments of the presentinvention may provide methods for performing a staining procedure (suchas a Gram staining procedure, for example) for a sample (see, element32, FIGS. 34 and 35, for example). In one embodiment, the methodcomprises step 103 for recording a first image of the sample 32. FIG. 1shows an exemplary first image of the sample 32 generated in a methodembodiment used to optimize a Gram staining process where Gram-positivebacteria as well as Gram-negative bacteria are present in an unstainedsample 32 that is fixed onto a surface 30 (defined by a microscopeslide, as shown in FIG. 34, for example). According to variousembodiments of the present invention, step 103 may comprise recording aseries of first images in different fields on the unstained sample 32.It should be understood that FIG. 1 shows an exemplary one line across afirst image. Because other method steps for applying a staining reagent(see step 104, FIG. 30, for example) may comprise colorizing theentities in the sample with a generally blue color, the first imageshown generally in FIG. 1 may be recorded in step 103 using a blue colorfilter disposed between the sample 32 and a camera device (such as a CCDcamera (see elements 5 and 8 of FIG. 32, for example).

The first image of FIG. 1 may serve as a “baseline” image from which afirst difference image (see FIG. 5, for example) may be produced (bycomparing a second image of the stained sample (FIG. 4) to the firstimage (FIG. 1). It should be understood for the purposes of this examplethat FIGS. 2 and 3 show the positions of stained and superficiallystained entities within the sample (corresponding, for example, toGram-positive and Gram-negative bacteria, respectively, present in thesample). For example, FIG. 2 is a symbolic illustration indicating thelocations of Gram-positive bacteria present in the sample along the sameone line image field described above with respect to FIG. 1. The word“Presence” on the Y-axis means in the context of FIG. 2 that afunctioning detector (such as an imaging system and/or camera device 8,for example) for detecting Gram-positive bacteria with spatialresolution would produce a signal at the X-positions indicated on theX-axis of FIG. 2. Furthermore, FIG. 3 is a symbolic illustrationindicating the locations of Gram-negative bacteria present in the samplealong the one line image field described above with respect to FIG. 1.The word “Presence” on the Y-axis means in the context of FIG. 3 that afunctioning detector (such as an imaging system, for example) fordetecting Gram-negative bacteria with spatial resolution would produce asignal at the X-positions indicated on the X-axis of FIG. 3.

As shown generally in FIG. 30 various sample preparation steps may beperformed prior to step 103 for recording one or more first images asshown in FIG. 1. For example, some method embodiments may comprise step101 for applying the sample or specimen to a surface (such as amicroscopic slide) and fixing the sample thereto. For example, in someembodiments, the sample 32 (see FIG. 34, for example) may be disposedand thermo-fixed (via the application of subtle heat, for example) ontomicroscope slide 30 defining a surface within a typical sampleattachment area 31. FIG. 34B is an exemplary side view of slide 30 witha sample 32 attached.

As described herein with respect to various system embodiments of thepresent invention, the prepared slide 30 may then be “snapped” into aflow channel housing (see generally FIGS. 34B and 34C) defining a slideaperture configured for receiving the slide 30 such that the sample 32is disposed substantially between the slide 30 and the flow channelhousing when the slide is disposed in the slide aperture. As describedfurther herein, the flow channel housing may comprise a substantiallytranslucent material such that an imaging system 5 (see FIG. 35, forexample) may be capable of recording images of the sample while thesample is disposed between the slide 30 and the flow channel housing.The flow channel housing may comprise a disposable body structured sothat the snapped-in microscope slide 30 and a cover glass, which may beintegrated into the body, may form a flow-through cell for theapplication of washing solutions (see step 102, for example),decolorizing reagents (see steps 107 and 112, for example) and/or theapplication of staining reagents (see step 104, for example). Theresulting flow-through cell (or flow channel, as described furtherherein) may comprise an inlet port and an outlet port for the washing,decolorizing, and staining reagents. One example of a completeddisposable flow channel is shown generally in FIG. 34D, and is shownoriented for positioning on an imaging system stage (such as amicroscope stage, for example) in FIG. 34E.

It should be understood that the various method steps for washing (step102), applying a staining agent (step 104), and applying a decolorizingagent (steps 107 and 112, for example) may be accomplished by a varietyof washing and/or fluid application techniques other than thosedescribed herein with respect to the flow chamber system embodimentsdepicted, for example in FIGS. 34D and 34E. For example, application ofwashing solutions, staining agents, and/or decolorizing agents may beaccomplished by a variety of known laboratory procedures including, butnot limited to: pipetting; aspiration; mixing; centrifuging; andcombinations of such processes.

Furthermore, as shown generally in FIG. 30, some method embodiments mayfurther comprise step 102 for washing the sample with a washing solutionprior to recording the first image of the sample. For example, step 102may comprise introducing a substantially colorless washing solution intoa flow channel or other space defined between the surface on which thesample 32 is operably engaged and a cover slip (and/or a flow channelhousing configured for receiving a microscope slide defining thesurface). The washing solution may be flushed through the flow channelin order to “condition” or “prime” the sample, by bringing it (and thebiological entities making up the sample) into contact with a liquid.The washing step 102 may be important for the later steps of comparingthe various images with each other (i.e. when generating differenceimages) because step 102 may ensure that the various entities within thesample properly and characteristically absorb and/or superficiallyabsorb the staining agent that may be applied, for example, in step 104(as described further herein). Furthermore, the washing step 102 mayalso advantageously fill the space between the sample and a cover slip(or flow channel housing) with the substantially colorless washing fluidwhich may, in turn, provide also for improved imaging conditions whenrecording images (see step 103, for example) of the sample.

As is known to persons skilled in the art of Gram staining, eachstaining step and each decolorizing step comprises a partial step ofwashing the sample with a washing solution such as water to remove anyexcess of staining dye or decolorizing reagent. For simplicity, we donot mention these partial steps and consider them as being an integralpart of a staining step and/or a decolorization step. Washing step 102,which is applied prior to any staining or decolorizing step, is the onlyexemption, and is mentioned therefore.

Referring again to FIG. 30, after a baseline first image (see FIG. 1,for example) of an unstained sample is recorded (in step 103, forexample) various method embodiments of the present invention may furthercomprise step 104 for applying a staining agent to the sample so as toprepare a stained sample comprising a plurality of stained entities. Theplurality of stained entities may include at least one of a plurality ofstained entities (wherein the staining agent is absorbed by the entity(i.e. Gram-positive bacteria, for example) and a plurality ofsuperficially stained entities (wherein the staining agent is merelypresent on an exterior surface of the entity (i.e. Gram-negativebacteria, for example)). As described herein with respect to varioussystem embodiments of the present invention, step 104 for applying thestaining agent may be accomplished by flushing the staining agentthrough a flow channel (defined in part by a surface with which thesample may be operably engaged) in order to stain the sample.Furthermore, as described herein, the staining agent applied in step 104may comprise a Gram stain. According to such embodiments, thesuperficially stained entities may comprise a plurality of Gram-negativeentities, and the stained entities may comprise a plurality ofGram-positive entities. In addition, it should be understood that thestaining agent applied in step 104 may comprise a variety of stainingagents that may be appropriate for preparing a sample for downstreamimaging and morphological examination. For example, the staining agentmay include, but is not limited to, a reagent comprising crystal violetand Gram's iodine.

After a staining agent is applied to the sample (in step 104, forexample), some method embodiments may further comprise step 105 forrecording a second image of the stained sample. An exemplary secondimage along the same one line image field described above with respectto FIG. 1 is shown, for example, in FIG. 4. FIG. 4 shows the locationsof all bacterial entities (Gram-positives as well as Gram-negatives, forexample) that are present along the one line. Depending on the detailedfeatures of the first image shown in FIG. 1, the “visibility” of theentities in FIG. 4 may be more or less optimal depending on thestructured background signal that may be present.

Referring again to FIG. 30, the method may further comprise step 106 forgenerating a first difference image (see FIG. 5) by comparing the secondimage (see FIG. 4, for example) to the first image (see FIG. 1, forexample) so as to determine a location of at least one of the stainedentities (if present) on the surface of the slide. The first differenceimage generated in step 106 (and shown, for example, in FIG. 5), showsthat the visibility of the bacteria or other entities present in thesample can be significantly enhanced according to various embodiments ofthe present invention by generating a first difference image. The firstdifference image may be produced in step 106 by an image processingcomputer 18 in communication with an imaging system comprising a camera8 (see FIG. 32, for example). For example, step 106 may be performed bythe image processing computer 18 performing an image processing step(such as image subtraction for example) between the second image (seeFIG. 4, for example) and the first image in (see FIG. 1, for example).By performing such an image processing step (such as image subtraction),many of the image features that are not influenced by a Gram stainingstep may be removed from the first difference image (as shown in FIG. 5,for example). As described herein with respect to the various systemembodiments of the present invention, a difference image signal (S) maybe generated using an image processing algorithm in the form:S=(J1−J2)/(J1+J2); wherein J1 and J2 refer to signal intensities inprimary and secondary images, respectively.

Therefore, only features related to the present entities (such asbacteria), and that are responsive to the staining procedure, are shownin the first difference image of FIG. 5, whereas the structuredbackground signal (generally visible in the first image of FIG. 1) isremoved. The first difference image (FIG. 5, for example) illustrates atechnical effect of one embodiment of the method of the presentinvention as it highlights and enhances visualization of the locationsof stained and/or superficially stained entities (such as Gram-positiveand Gram-negative bacteria, for example) on a slide. This enhancementeffect is particularly valuable for possible later visual inspection ofthe images (such as a morphology analysis (see step 116, for example)).

Various method embodiments may also comprise step 107 for applying adecolorizing agent to the stained sample so as to prepare a partiallydecolorized sample wherein at least a portion of the staining reagent isremoved from the superficially stained entities. According to oneexemplary embodiment, FIG. 6 shows a third image that has been recordedafter performing a partial decolorization (see step 107, for example) ofthe sample, in this case a decolorization by approximately 35% of totaldecolorization. According to various embodiments, step 107 may alsocomprise controlling the degree of decolorization that may be performed,e.g., by timing the duration of flushing the space in between the slide2 and the cover slip 3 (see FIG. 31, for example) with a decolorizingagent such as acid alcohol. As can be seen from the plot in FIG. 6, thesignals corresponding to superficially stained entities (comprisingGram-negative bacteria, for example) at X-positions 50 and 80 may bedecreased somewhat by the addition of the decolorizing agent in step107. A certain percentage of decolorization may also be apparent for thestained entities (corresponding to Gram-positive bacteria, for example)that may also be present in the sample, but the decolorization ofstained entities is generally much less pronounced, compared to thedecolorization of superficially stained entities (corresponding toGram-negative bacteria, for example). FIG. 30 also shows step 108 forrecording a third image of the partially decolorized sample. Step 108generally results in a third image shown, for example, in FIG. 6.

Some method embodiments, as shown generally in FIG. 30, may furthercomprise step 109 for generating a second difference image (see FIG. 7,for example) by comparing the third image (see FIG. 6, for example) tothe second image (see FIG. 4, for example) so as to determine a locationof at least one of the superficially stained entities on the surface ofthe slide 2 (see FIG. 31, for example). FIG. 7 depicts such an exemplarysecond difference image showing only signals related to superficiallystained entities (corresponding to Gram-negative bacteria, for example).As described further herein with respect to various system embodimentsof the present invention, the second difference image shown in FIG. 7may be generated by an image processing computer 18 (in communicationwith a CCD camera 8, for example). For example, the image processingcomputer 18 may be configured for generating the second difference image(FIG. 7, for example) by performing an image subtraction step(subtracting the third image (FIG. 6) from the second image (FIG. 4, forexample) to highlight those entities within the sample that have beenmost affected (i.e. decolorized) by the addition of the decolorizingagent in step 107). In Gram-staining embodiments, step 109 may revealand/or highlight the positions of Gram-negative bacteria, which respondmost to the decolorization procedure. Step 109 for generating the seconddifference image also provides the technical effect of allowing a user(and/or the image processing computer 18) to quantitatively and/orqualitatively compare the signals for superficially stained entities(i.e. Gram-negative bacteria) in the plots of FIG. 4 and FIG. 6 and toperform step 110 for determining the current degree of decolorizationachieved during the partial decolorization step (step 107, for example).

Thus, as shown in FIG. 30, step 109 for generating the second differenceimage also provides a basis for comparison to allow for the performanceof step 111 for estimating the necessary further decolorization time toobtain an optimized decolorization of the sample. More particularly, insome method embodiments, step 111 may comprise analyzing the seconddifference image (see FIG. 7, for example) to determine an exposure timeduring which the decolorizing agent should be applied to the partiallydecolorized sample to substantially decolorize the superficially stainedentities (i.e. Gram-negative bacteria, for example) withoutsubstantially decolorizing the stained entities (i.e. Gram-positivebacteria, for example). Step 111 may comprise quantitatively estimatingan exposure time for subsequent step 112 to substantially decolorize thesuperficially stained entities by performing a ratio calculation basedat least in part on the exposure time that resulted in the level ofdecolorization in the superficially stained entities that is apparentfrom viewing the “blue intensity” of the second difference image shown,for example, in FIG. 7.

Some method embodiments may further comprise step 112 for applying thedecolorizing agent (such as acid alcohol, for example) to the partiallydecolorized sample (for the exposure time determined in step 111) so asto prepare a substantially decolorized sample wherein the superficiallystained entities (i.e. Gram-negative bacteria, for example) aresubstantially decolorized and wherein the stained entities(Gram-positive bacteria, for example) are not substantially decolorized.As shown in FIG. 30, the method may also comprise step 113 for recordinga fourth image (see FIG. 8, for example) of the substantiallydecolorized sample. FIG. 8 shows a fourth image obtained, for example,after applying the final decolorization step (step 112). As shown inFIG. 8, only signals corresponding to stained entities (i.e.Gram-positive bacteria) remain detectable at X-positions 30 and 90 onthe slide surface.

Referring again to FIG. 30, various method embodiments may also comprisestep 114 for generating a third difference image (see FIG. 9, forexample, showing an exemplary third difference image produced by animage processing computer 18 (see FIG. 32)). The third difference imagemay be produced by comparing the fourth image (see FIG. 8) with thesecond image (see FIG. 4, for example). In some embodiments, the thirddifference image of FIG. 9 may be produced by performing an imagesubtraction procedure to “subtract” the signals detected in the fourthimage (FIG. 8) from those detected in the second image (FIG. 4). Asshown in FIG. 9, the third difference image may clearly depict thelocation of at least one of the superficially stained entities (i.e.Gram-negative bacteria, for example) on the surface. In the exemplaryplot of the third difference image shown in FIG. 9, the locations ofGram-negative bacteria are depicted at locations 40 and 80 on the slidesurface.

In order to confirm the locations (i.e. X-positions, for example) ofstained entities (i.e. Gram-positive bacteria) on the slide surface,some method embodiments may further comprise step 115 for generating afourth difference image (see FIG. 10) by comparing the fourth image(FIG. 8) to the first image (FIG. 1). As shown in FIG. 10, the fourthdifference may depict a location of at least one of the stained entitieson the surface of the slide. In some embodiments, step 115 may beperformed by an image processing computer 18 in communication with a CCDcamera 8 (or other imaging system component) to subtract the signal plotin FIG. 1 from the signal plot in FIG. 8. The difference image (FIG. 10,for example) shows the signals (and corresponding locations) ofGram-positive bacteria.

In some method embodiments, the third and fourth difference images (inFIGS. 9 and 10, respectively) may be used to perform a more detailedmorphology analysis (see step 116) for the purpose of furtherclassification of all entities (i.e. Gram-positive and Gram-negativebacteria, for example) found in the sample 32. Step 116 may beperformed, for example, using an image processing computer 18 having anintegrated memory device (not shown) configured for storing a library ofknown entities, in order to aid a user in classifying the variousentities (i.e. Gram-negative and Gram-positive bacteria, for example)whose locations have been determined and/or confirmed by the variousmethod steps shown, for example, in steps 101-115 of FIG. 30. Forexample, step 116 may comprise analyzing a morphology of thesuperficially stained entities (i.e. Gram-negative bacteria).Furthermore, in some embodiments, step 116 may also comprise analyzing amorphology of the stained entities (i.e. Gram-positive bacteria).

FIGS. 11-19 show various images generated according to the variousmethod steps of the present invention (see FIGS. 30 and 36, generally)in an exemplary case wherein only Gram-positive bacteria are present inthe sample 32. Specifically, FIGS. 11-13 correspond fully to FIGS. 1-3,respectively, showing the first image, the locations of Gram-positivebacteria (at X-positions 30 and 90 on the slide surface, for example),and the locations of Gram-negative bacteria (none). Furthermore, FIG. 14depicts a second image taken after the application of a staining reagent(see step 104, for example), showing only signals corresponding toGram-positive bacteria.

FIG. 15 shows a first difference image with background-free signals(obtained, for example, by subtracting the “background” signal of FIG.11 from the post-stain image of FIG. 14 in order to highlight thelocations of the Gram-positive bacteria. Furthermore, FIG. 16 shows athird image obtained after application of a decolorizing agent to obtainpartial decolorization of the sample. FIG. 17 is a second differenceimage, which would, in some cases, show the signals from Gram-negativebacteria. As expected in this case (from the known data shown in FIG.13) no signals are present. This result shows that the formalapplication of the method steps of the present invention (see FIGS. 30and 36, for example) may provide correct information about the presenceor absence of Gram-negative bacteria without significant input from anoperator. FIG. 18 is a third difference image, which also would indicatethe presence of Gram-negative bacteria, if present. Finally, FIG. 19shows a fourth difference image showing the signals and locations ofGram-positive bacteria in excellent contrast so as to allow for a morecomplete morphology analysis of the Gram-positive bacteria (see step116, FIG. 30, for example).

FIGS. 20-29 show various images generated according to the variousmethod steps of the present invention (see FIGS. 30 and 36, generally)in an exemplary case wherein only superficially-stained entities (i.e.Gram-negative bacteria) are present in the sample 32. Specifically,FIGS. 20-22 correspond fully to FIGS. 1-3, respectively, showing thefirst image, the locations of Gram-positive bacteria (none), and thelocations of Gram-negative bacteria (at X-positions 50 and 80 on theslide surface, for example).

FIG. 23 shows a second image, showing only signals from Gram-negativebacteria after an application of a staining reagent. FIG. 24 shows afirst difference image with background-free signals, clearlyillustrating the locations of Gram-negative bacteria at X-positions 50and 80. As described herein with respect to various method and systemembodiments, each of the difference images (FIGS. 24, 26, 28 and 29, forexample) may be generated by an image processing computer 18 (see FIG.32) configured for performing an image subtraction procedure using thevarious images provided by a CCD camera 8 or other imaging systemcomponent. FIG. 25 shows a third image obtained after partialdecolorization of the sample, which may be achieved via the applicationof a decolorizing agent (see step 107, FIG. 30, for example).

FIG. 26 shows a second difference image, which would show the signalsfrom Gram-negative bacteria. As expected in this case, signals arepresent. This result shows again that the formal application of themethod steps according to various embodiments of the present invention,achieves the technical effect of providing correct information about thepresence or absence of Gram-negative bacteria in a sample 32 without theneed for substantial input from an operator. FIG. 27 is a fourth imageobtained after final decolorization. As expected, no signals from thesuperficially-stained Gram-negative bacteria are detectable in thisimage. FIG. 28 is a third difference image, showing the clear presenceand location of Gram-negative bacteria on the slide surface. Finally,FIG. 29 is a fourth difference image, which would indicate the presenceof Gram-positive bacteria, were such present in the sample. As expectedin this case (see FIG. 21), no signals from Gram-positive bacteria aredetected.

FIG. 36 shows a schematic flow chart of a method, according to analternate embodiment of the present invention for staining a sample. Themethod first comprises step 201 for operably engaging a sample with asample stage 1 of an imaging system (see generally, FIG. 35). Step 201may be accomplished, in some embodiments, as shown generally in FIG. 34.For example, FIG. 34 is an illustration of the sample installationprocedure that may be used in one embodiment of the present invention.In FIG. 34A, the sample 32 is disposed and thermo-fixed onto an ordinarymicroscope slide 30 within a typical sample attachment area 31. FIG. 34Bis a side view of slide 30 with a sample 32 attached. The prepared slide30 is then “snapped” into a disposable body with the one surface havingthe sample 32 operably engaged therewith (see generally, FIG. 34C). Thedisposable body is structured so that the snapped-in microscope slideand a cover glass, which is integrated into the body, form a flowchamber 20 for applying at least one of the staining and decolorizingreagents to the sample 32. As shown in FIG. 35, the flow chamber 20 maydefine a reagent inlet 22, and a reagent outlet 23 for “washing” thedecolorizing and staining reagents through the flow chamber 20 and pastthe sample 32. One embodiment of a completed disposable flow chamber isshown in FIG. 34D. The flow chamber 20 may be inverted for positioningon a microscope stage 1 (see FIGS. 34E and 35).

As shown in FIG. 36, the various method embodiments may also comprisestep 202 for recording images of the sample using the imaging systembefore and after an application of a staining reagent and adecolorization reagent to the sample. For example, step 202 may compriserecording images of the sample at various stages of the staining and/ordecolorization processes described herein. The images recorded in step202 may include, but are not limited to: an image of the unstainedsample (see FIG. 1, for example); an image of the stained sample (seeFIG. 4, for example); and an image of a partially decolorized sample(see FIG. 6, for example).

The method may further comprise step 203 for generating a differenceimage based at least in part on a comparison of the images of the samplerecorded before and after the application of the staining reagent andthe decolorization reagent to the sample. For example, step 203 maycomprise, in some embodiments, generating a second difference image (seeFIG. 7, for example) by subtracting the image signals of the image ofthe partially decolorized sample (see FIG. 6, for example) from theimage signals present in the image of the stained sample (see FIG. 4,for example).

Referring again to FIG. 36, various method embodiments of the presentinvention may also comprise step 204 for correcting the application ofthe staining reagent and/or the decolorization reagent based at least inpart on the difference image generated in step 203. For example, step204 may comprise determining quantitatively the degree of decolorizationapparent in the sample (and/or in the superficially-stained entitiesincluded therein) by analyzing the difference image of step 203. Forexample, the measurable “blue intensity” of FIG. 7 generally indicatesthe quantitative amount of decolorization achieved during theapplication of a decolorizing agent. Given a known exposure and/or“flushing” time, step 204 may comprise determining a corrected and/oradditional exposure time that would be expected to remove the remainingstaining reagent from the superficially stained entities present in thesample. The method may further comprise step 205 for reapplying at leastone of the staining reagent and the decolorization reagent according tothe various parameters (such as exposure and/or flush time) calculatedin the correcting step 204. Finally, as shown in FIG. 36, such methodembodiments may further comprise generating a final image (see FIG. 8,for example) of the sample so as to differentiate at least one target ofinterest (i.e. Gram-positive entities) in the sample that may berendered discernible by the reapplying step 205.

Various embodiments of the present invention may also provide systemand/or apparatus embodiments for performing an optimized stainingprocedure (such as a Gram stain procedure) for a sample 32. For exampleone system embodiment, shown generally in FIGS. 32 and 35 comprises aflow chamber 20 defining a channel in fluid communication with a supplyof a staining agent 11 and a supply of a decolorizing agent 12, whereinthe flow chamber comprises a surface 31 configured for operably engagingthe sample 32 therewith (see FIGS. 34A-34E, for example). In some systemembodiments, the flow chamber may be defined between a slide 2 and coverglass 3 having a spacer 4 operably engaged therebetween to define a flowchamber (as shown generally in FIG. 31 and as described further herein).

As shown generally in FIGS. 34 and 35, the flow chamber 20 may comprisea slide 30 defining the surface 31 for operably engaging the sample 32therewith. The flow chamber 20 may also comprise a flow channel housingdefining a slide aperture adapted for receiving a standard slide (seeFIGS. 34B-34C showing the slide being operably engaged with the flowchannel housing) such that the sample 32 is disposed substantiallybetween the slide 30 and the flow channel housing when the slide isdisposed in the slide aperture. Furthermore, the flow channel housingmay comprising a substantially translucent material (such as anintegrated glass and/or polycarbonate cover glass 21) such that theimaging system (and/or a lens 5 thereof) may be capable of recordingimages of the sample 32 while the sample 32 is disposed between theslide 30 and the flow channel housing.

The flow chamber 20 (and/or a systems controller 19 portion of thesystem in communication therewith) may cooperate with a fluidics system(comprising, for example, various appropriate pumps 14, valves 15, andone/or more combining valves 16, as shown in FIG. 32, for example) thatis configured for applying the staining agent to the sample 32 (via areagent inlet 22, for example) so as to prepare a stained samplecomprising a plurality of stained entities. As described herein withrespect to various method embodiments, the plurality of stained entitiesmay include at least one of a plurality of stained entities(Gram-positive entities, for example) and a plurality of superficiallystained entities (Gram-negative entities, for example). The fluidicssystem 14, 15, 16 (in cooperation with the flow chamber 20) may befurther configured for applying the decolorizing agent to the stainedsample (via the reagent inlet 22, for example) so as to prepare apartially decolorized sample wherein at least a portion of the stain isremoved from the superficially stained entities. In some system and/orapparatus embodiments, the fluidics system 14, 15, 16 (in cooperationwith the flow chamber 20 and/or a systems controller 19) may be furtherconfigured for applying the decolorizing agent 12 to the partiallydecolorized sample for a determined exposure time (calculated, forexample, in step 111 of FIG. 30, for example) so as to prepare asubstantially decolorized sample wherein the superficially stainedentities are substantially decolorized and wherein the stained entitiesare not substantially decolorized by the application of the decolorizingagent. Furthermore, and as shown generally in FIG. 32, various systemembodiments may comprise reservoirs 10, 11, 12, and 13, containingwashing, staining, decolorizing, and counter-staining reagents,respectively. The systems controller 19 may be configured for directingthese reagents towards reagent inlet 6 of the flow-through disposable(i.e. the flow chamber 20) via appropriate pumps 14, valves 15, andone/or more combining valves 16. Reagents that are flushed through theflow chamber 20 may be collected in a waste reservoir 17 provided insome system embodiments.

System and apparatus embodiments of the present invention may alsocomprise an imaging system (and/or an objective lens 5 thereof) disposedadjacent to the flow chamber 20 such that the sample 32 is positionedwithin a field of view of the imaging system. As shown in FIG. 32, theimaging system may comprise: an objective lens 5; an imaging receiver orcamera device 8 such as a CCD camera configured to be capable ofrecording images of the sample while the sample 32 is disposed betweenthe slide 30 and the flow channel housing 20; and an actuator device(such as an XYZ-translation mechanism 9, for example) operably engagedwith the camera device 8 and configured for adjusting a position of thecamera device 8 relative to the flow chamber 20. It should be understoodthat, in alternate system embodiments, the system may comprise anactuator mechanism (such as an XYZ-translation mechanism) operablyengaged with the stage 1 (see FIG. 32) and configured for adjusting aposition of the flow chamber 20 relative to the camera device 8. In someembodiments, the camera device 8 may be in communication with an imageprocessing computer 18 (as described further herein), which may be incommunication with a systems controller 19. Furthermore, the systemscontroller 19 may also be in communication with a control input of theXYZ-translation mechanism 9 to move objective lens 5 to different fieldsof interest on the sample 32, and to perform auto-focus operationsrelative to the sample 32.

Because the slide 30 and/or flow chamber 20 are operably engaged with astage 1 of the imaging system, the components of the imaging system(such as the imaging receiver 8) may be configured for monitoring thesample 32 (in real time), before, after, and/or during an application ofat least one of the staining agent and the decolorizing agent. Theimaging system (and/or the image processing computer 18 thereof) may befurther configured for generating a difference image (see FIG. 7, forexample) by comparing at least one image of the sample 32 obtainedbefore a first application of the decolorizing agent (see FIG. 4, forexample) and at least one image of the sample obtained after the firstapplication of the decolorizing agent (see FIG. 6, for example).

The imaging system (and/or an image processing computer 18 includedtherein) may be further configured, in some embodiments, to determine anexposure time (see step 111, FIG. 30, for example) for a secondapplication of the decolorizing agent based at least in part on thedifference image (FIG. 7) so as to substantially decolorize thesuperficially stained entities without substantially decolorizing thestained entities such that the stained entities may be more readilydiscerned from the superficially stained entities. Various systemembodiments may also comprise a controller device (such as the systemscontroller 19, shown in FIG. 32, for example) in communication with theimaging system 8, 9, 18 and the fluidics system 14, 15, 16. Thecontroller device may be configured for controlling the application ofat least one of the staining agent and the decolorizing agent based atleast in part on the images generated by the imaging system (comprising,for example, a camera device 8).

In some system embodiments, the imaging system (and/or componentsthereof (such as the image processing computer 18)) may be configuredfor performing one or more of the method steps outlined generally inFIGS. 30 and 36. For example, in one embodiment, the imaging system(and/or a camera device 8 thereof) may be configured for performing step103 for recording a first image (see FIG. 1) of the sample 32. Theimaging system may be further configured for recording a second image(see FIG. 4) of the stained sample after applying the staining agent tothe sample using the flow chamber 20.

Furthermore, the imaging system (and/or an image processing computer 18included therein) may be further configured for generating a firstdifference image (see FIG. 5, for example) by comparing the second image(FIG. 4) to the first image (FIG. 1) so as to determine a location of atleast one of the stained entities on the surface 31 of the slide 30. Theimaging system (and/or the camera device 8 thereof) may be furtherconfigured for recording a third image (FIG. 6, for example) of thepartially decolorized sample after applying the decolorizing agent (seestep 107, FIG. 30) using the flow chamber 20. The imaging system (and/orthe image processing computer 18) may be further configured forgenerating a second difference image (see FIG. 7) by comparing the thirdimage (FIG. 6) to the second image (FIG. 4) so as to determine alocation of at least one of the superficially stained entities. Finally,in some system embodiments, the imaging system (and/or the imageprocessing computer 18 in cooperation with the systems controller 19)may be further configured for performing step 111 for analyzing thesecond difference image (FIG. 7) to determine an exposure time duringwhich the decolorizing agent is applied to the partially decolorizedsample to substantially decolorize the superficially stained entitieswithout substantially decolorizing the stained entities.

According to various system embodiments of the present invention, theimaging system (including various image processing computer 18 and/orcamera device 8 components) may be further configured for performingvarious method steps including, but not limited to: step 113 forrecording a fourth image of the substantially decolorized sample (seeFIG. 8, for example); step 114 for generating a third difference image(FIG. 9) by comparing the fourth image to the second image; and step 115for generating a fourth difference image (FIG. 10) by comparing thefourth image to the first image. As described herein with respect tovarious method embodiments of the present invention, the thirddifference image (see FIG. 9, for example) may depict the location of atleast one of the superficially stained entities (i.e. Gram-negativeentities, for example). Furthermore, the fourth difference image (seeFIG. 10, for example) may depict a location of at least one of thestained entities (i.e. Gram-positive entities, for example).

As shown in FIG. 32, the imaging system provided in various systemembodiments of the present invention may comprise an image processingcomputer 18 configured for generating difference images that mayinclude, but are not limited to: the first difference image (FIG. 5, forexample), the second difference image (FIG. 7, for example), the thirddifference image (FIG. 9, for example), and the fourth difference image(FIG. 10, for example). In such system embodiments, the image processingcomputer 18 may be configured for generating one or more of the variousdifference images using processes that may include, but are not limitedto: image subtraction; image addition; image ratio calculation; andcombinations of such image processing routines. For example, in someembodiments, the image processing computer 18 (and/or the systemscontroller 19, in some embodiments) may be configured for generating adifference image signal (S) using an image processing algorithm in theform: S=(J1−J2)/(J1+J2); wherein J1 and J2 refer to signal intensitiesin primary and secondary images, respectively.

FIG. 31 is a schematic depiction of a system according to one embodimentof the present invention. The biological sample is disposed and fixedonto a microscope slide 2, which may be positioned on the XY-stage 1 ofa microscope and is imaged through a microscope objective lens 5 towardsan imaging receiver such as a CCD camera (or other camera device 8 (seeFIG. 32). A cover glass 3 is held at a selected distance from microscopeslide 2 by means of a spacer 4 having an appropriate thickness. Thespacer 4 may form a flow channel between slide 2 and cover glass 3 witha reagent inlet 6 on one side and a reagent outlet 7 on another side.The spacer 4 may comprise an elastomeric substance configured to providesubstantially fluid-tight engagement between the spacer 4 and the slide2 as well as between the spacer 4 and the cover glass 3 such that aparallel flow chamber is formed. The effective thickness of spacer 4 maybe selected such that sharp images of the biological sample onmicroscope slide 2 may be obtained by the camera device 8 (or otherimaging system component) even if the objective lens 5 has amagnification of 100×.

FIG. 33 shows an example of a snap-in flow-through disposable suitableto practice various method and/or system embodiments of the presentinvention. FIG. 33A shows the disposable as seen from above, i.e. fromwhere objective lens 5 shown in FIGS. 31 and 32 would be located. Inother embodiments, the objective lens 5 may be disposed below thedisposable. In such embodiments, FIG. 33A would represent a view from“below”. FIG. 33A shows the preformed disposable body 20, which in thisexample has a cover glass 21 integrated therein. Also shown is a reagentinlet 22 as well as a reagent outlet 23 that may be in fluidcommunications with the reservoirs 10, 11, 12, 13 shown in FIG. 32, forexample). FIG. 33B shows a side view of the disposable depicting apreformed disposable body 20, an integrated cover glass 21, a reagentinlet 22, and a reagent outlet 23. After operably engaging the preparedmicroscope slide 30 with the body 20, a flow-through chamber 28 withconnector channels 26 and 27 to inlet 22 and outlet 23, respectively, isformed. In some embodiments, the disposable body 20 may comprise acorner “lip” feature 25, which may allow the slide 24 to be releasably“snapped-in” to the disposable body to form the flow-through chamber 28between the slide 30 and body 20. FIG. 33C shows a rear view of thedisposable depicting, in particular, one exemplary embodiment ofconnector channels 26 and 27 that may be defined between theflow-through chamber 28 and at least one of the reagent inlet 22 and thereagent outlet 23.

FIG. 34 illustrates an exemplary procedure for operably engaging asample 32 with a surface (such as a sample attachment area 31 of aconventional microscope slide 30. In FIG. 34A, the sample 32 to beevaluated may be disposed and thermo-fixed onto an ordinary microscopeslide 30 within a typical sample attachment area 31. FIG. 34B is a sideview of slide 30 with a sample 32 attached. The prepared slide is then“snapped” into a disposable body with the one side carrying the sample32 (see FIG. 34C). The disposable body is structured so that thesnapped-in microscope slide and a cover glass, (which is integrated intothe body) may form a flow channel for the various washing, staining,decolorization, and/or counterstaining reagents that may be used invarious embodiments of the present invention. As described herein, theflow chamber may comprise a reagent inlet port and a reagent outlet portfor washing reagents into and out of the flow chamber. The completeddisposable is shown in FIG. 34D, and is finally turned upside down forpositioning on a microscope stage (see FIG. 34E).

FIG. 35 shows a completed disposable, positioned on a microscope andconnected with a liquid handling system to perform the various washing(see step 102), staining (see step 104), and/or decolorization (seesteps 107 and 112) procedures according to various embodiments of thepresent invention. In some embodiments, fluid communication between theflow chamber and a liquid handling system (see elements 14, 15 and 16 ofFIG. 32, for example) may be established by connector pipes 33 and 34that may be configured for holding the disposable flow chamber 20 inplace relative to the imaging system (and/or an objective lens 5thereof) and for providing substantially fluid-tight connection to thereagent inlet 22 and the reagent outlet 23 via pressure fittings 35 and36, respectively. The connector pipes 33, 34 may, in some embodiments,comprise a substantially stiff polymeric substance configured for urgingthe disposable flow chamber 20 generally downward and into engagementwith a stage 1 of the imaging system.

It should be understood that the various system embodiments describedherein with respect to FIGS. 31-35 may be configured for performing thevarious method steps that have been described in connection with thevarious images shown in FIGS. 1-29 and that are summarized in FIGS. 30and 36. For example, referring to the system embodiment shown in FIG.32, the systems controller 19 may be configured for controlling theautomated staining process (see step 104, for example) anddecolorization processes (see steps 107 and 112). The systems controller19 may also be configured for communicating with the image processingcomputer 18 and the actuator device 9 (comprising an XYZ-translationmechanism, for example) to determine when to perform an auto-focusoperation, when to record an image, when to move to a new field ofinterest, and when image processing steps (such as image subtraction,for example) should be performed to generate various difference images.In some embodiments, the image processing computer 18 may automaticallyproduce optimized images showing either Gram-positive bacteria orGram-negative bacteria. Such images may be stored by the imageprocessing computer 18 (in a memory device integrated therein, forexample) and made available to a lab technician, microbiologist, orother user so as to enable the user to perform a manual morphologyanalysis and/or to identify various entities that may be present in agiven sample. For example, in some embodiments, the image processingcomputer 18 may comprise a user interface (such as a high-resolutiondisplay (not shown)) configured for generating a visual depiction of theoptimized images of Gram-positive bacteria or Gram-negative bacteriawithin the sample such that a user may perform step 116 for analyzing amorphology of the sample entities without the need to remove the samplefrom a microscope stage 1 or other analysis field relative to an imagingsystem. In other embodiments, the image processing computer 18 maycomprise a memory device containing an image data base including avariety of known microorganisms (and characteristic stained imagescorresponding thereto). The image processing computer 18 may thenexecute an automated bacterial identification procedure, andsubsequently offer the result to a user via a user interface. The usermay then accept the automated identification output, or use theautomated result as a starting point for a follow-up manualconfirmation. In these various system embodiments, the sample 32 mayremain on the sample stage 1 of the imaging system, so that the user cancompare the various difference images generated by the image processingcomputer 18 with the unprocessed actual image of an optimally-stainedsample.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

What is claimed is:
 1. A method for performing a staining procedure fora biological sample, the method comprising: operably engaging a samplewith a sample stage of an imaging system; recording one or more imagesof the same using the imaging system before and after an application ofa staining reagent and a decolorizing reagent to the sample; generatinga difference image based at least in part on a comparison of the imagesof the sample recorded before and after the application of the stainingreagent and the decolorization reagent to the sample; and correcting theapplication of the staining reagent and the decolorization reagent basedat least in part on the difference image by analyzing the differenceimage to determine an exposure time for application of thedecolorization reagent.
 2. The method according to claim 1, furthercomprising: reapplying at least one of the staining reagent and thedecolorization reagent according to the correcting step and generating afinal image of the sample so as to differentiate at least one target ofinterest in the sample.
 3. The method according to claim 1, furthercomprising washing the sample with a washing solution prior to recordingan image of the sample.
 4. The method according to claim 1, wherein theapplication of the staining agent is designed to prepare a plurality ofstained entities and/or a plurality of superficially stained entities.5. The method according to claim 4, further comprising analyzing amorphology of the superficially stained entities, if present.
 6. Themethod according to claim 1, further comprising analyzing a morphologyof the stained entities, if present.
 7. The method according to claim 4,wherein the staining agent comprises a Gram stain and wherein thesuperficially stained entities, if present, comprise a plurality ofGram-negative bacteria, and wherein the stained entities, if present,comprise a plurality of Gram-positive bacteria.
 8. A system forperforming a staining procedure for a biological sample, the systemcomprising: a flow chamber defining channel in fluid communication witha supply of a staining agent and a supply of a decolorizing agent; afluidics system in fluid communication with the flow chamber, the supplyof the staining agent, and the supply of the decolorizing agent; animaging system configured for recording at least one image of the samplebefore, during, and/or after the application of at least one of thestaining agent and the decolorizing agent; and a controller device incommunication with the imaging system and the fluidics system, thecontroller device being configured to control the application of atleast one of the staining agent and the decolorizing agent, wherein theimaging system is further configured to determine an exposure time foran application of the decolorizing agent.
 9. The system according toclaim 8, wherein the flow chamber is adapted for receiving a slidedefining a surface for operably engaging the sample therewith andwherein the flow chamber comprises a flow channel housing defining aslide aperture configured for receiving the slide such that the sampleis disposed substantially between the slide and the flow channel housingwhen the slide is disposed in the slide aperture, the flow channelhousing comprising a substantially translucent material such that theimaging system is capable of recording the at least one image of thesample while the sample is disposed between the slide and the flowchannel housing.
 10. The system according to claim 8, wherein theimaging system comprises: a camera device configured for recording theat least one image of the sample while the sample is disposed betweenthe slide and the flow channel housing; and an actuator device operablyengaged with the camera device and configured for adjusting a positionof the camera device relative to the flow chamber.
 11. The system ofclaim 8, wherein the fluidics system is configured for applying thestaining agent to the sample so as to prepare a stained samplecomprising a plurality of stained entities and/or a plurality ofsuperficially stained entities, and the fluidics system is furtherconfigured for applying the decolorizing agent to the stained sample soas to prepare a partially decolorized sample wherein at least a portionof the stain is removed from the stained sample.
 12. The systemaccording to claim 11, wherein the fluidics system is further configuredfor cooperating with the flow chamber for applying the decolorizingagent to the partially decolorized sample for a determined exposure timeso as to prepare a substantially decolorized sample wherein thesuperficially stained entities are substantially decolorized if present,and wherein the stained entities are not substantially decolorized, ifpresent.
 13. The system according to claim 8, wherein the imaging systemcomprises an image processing computer configured for analyzing the atleast one image.
 14. The system according to claim 13, wherein the imageprocessing computer is configured for generating at least one differenceimage using processes selected from the group consisting of: imagesubtraction; image addition; image ratio calculation; and combinationsthereof.
 15. The system according to claim 11, wherein the stainingagent comprises a Gram stain and wherein the superficially stainedentities comprise a plurality of Gram-negative bacteria, and wherein thestained entities comprise a plurality of Gram-positive bacteria.